Does Demethylation of the Inactive X Contribute to Lupus in Women?

非活性 X 的去甲基化是否会导致女性狼疮？

• 批准号: 8597403
• 负责人: BRUCE C. RICHARDSON
• 金额: --
• 依托单位: VETERANS HEALTH ADMINISTRATION
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-01 至 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8597403
• 关键词: Affect; Animal Model; Autoantibodies; Autoimmune Diseases; Autoimmunity; B-Lymphocytes; CD4 Positive T Lymphocytes; CXCR3 gene; Cell physiology; Cells; DNA; DNA Methylation; DNA Sequence; Development; Disease; Epigenetic Process; Estrogens; Female; Flare; Frequencies; Gene Expression; Genes; Gonadal Steroid Hormones; Grant; Human; Immunoblotting; Individual; Klinefelter' s Syndrome; Link; Lupus; Methylation; MicroRNAs; Military Personnel; Modeling; Modification; Molecular; Mus; Pathogenesis; Predisposition; Prevalence; Production; Relative (related person); Reporting; Severities; Severity of illness; Signal Transduction; Systemic Lupus Erythematosus; T-Lymphocyte; TNFSF5 gene; Testing; Time; Transcript; Transfection; Turner' s Syndrome; UDP-N-acetylglucosamine-peptide beta-N-acetylglucosaminyltransferase; United States; Veterans; Woman; Work; X Chromosome; base; chemokine receptor; demethylation; design; effective therapy; environmental agent; inhibitor/antagonist; insight; lupus-like; male; men; mortality; new therapeutic target; novel; novel therapeutic intervention; novel therapeutics; overexpression; public health relevance

DESCRIPTION (provided by applicant): Systemic lupus erythematosus is a sometimes fatal autoimmune disease primarily affecting women. Estrogen contributes to disease severity, but does not completely explain the female predilection. Recent reports that men with Klinefelter's Syndrome (XXY) develop lupus at the same rate as women, while women with Turner's Syndrome (XO) are protected, suggests that having 2 X chromosomes is important for disease development. However, how a second X chromosome, which is largely inactivated, contributes to lupus in women is unclear. Evidence accumulated over more than 20 years indicates that lupus develops when genetically predisposed people are exposed to environmental agents that cause DNA demethylation in T cells, and that demethylated female T cells are sufficient to cause lupus-like autoimmunity. Work from the Richardson lab has shown that the B cell costimulatory molecule CD40L, encoded on the X chromosome and known to be overexpressed on lupus T cells, is overexpressed on T cells from women but not men with lupus because the gene on the inactive female X demethylates to cause biallelic expression, while men can only transcribe from one gene. Preliminary evidence suggests that demethylated T cells from women also overexpress other X chromosome genes including the chemokine receptor CXCR3, the signaling regulator OGT, and 6 microRNAs. Based on these observations we hypothesize that demethylation of genes on the inactive X predisposes women to lupus. We will test this hypothesis by: 1. Using high throughput DNA sequencing to compare methylation of CXCR3, OGT, the 6 miRNA genes and other X chromosome genes in CD4+ T cells from healthy men and women, with and without treatment with DNA methylation inhibitors, 2. Using high throughput DNA sequencing to compare methylation of CXCR3, OGT, the 6 miRNA genes and other X chromosome genes in CD4+ T cells from men and women with lupus matched for disease activity, 3. Determining the consequences of OGT overexpression on CD4+ T cell gene expression using transfections, microarrays and immunoblotting, and identify the miRNA target transcripts using Argonaute pulldowns followed by array analyses or sequencing, and 4. Determining if overexpressing CD40L, CXCR3, OGT or X chromosome miRNAs in a recently developed murine lupus model increases the severity of lupus in males to approximate the severity in females. We anticipate that these studies will confirm that women are predisposed to lupus in part because they can overexpress more genes than men, identify the molecules responsible, and thereby identify new therapeutic targets for women with lupus. PUBLIC HEALTH RELEVANCE: According to a recent estimate, SLE afflicts more than 130,000 individuals in the United States. Of these, nearly 90% are women, and women now comprise a significant number of military veterans. Current estimates reveal that the mortality rate of women with lupus is as high as 17.6 per million per year, indicating that current therapies are still imperfect. The design of more effective treatments will require a clear understanding of lupus pathogenesis. The studies described in this applicatiion will characterize molecular mechanisms predisposing women to lupus and thus may contribute to the design of safer and more effective therapies for this disease, and possibly for other autoimmune diseases that afflict our female veterans.

描述（由申请人提供）： 系统性红斑狼疮是一种有时致命的自身免疫性疾病，主要影响女性。雌激素会导致疾病的严重程度，但并不能完全解释女性的偏好。最近的报告称，患有克兰费尔特综合症 (XXY) 的男性患狼疮的速度与女性相同，而患有特纳综合症 (XO) 的女性则受到保护，这表明拥有 2 条 X 染色体对于疾病的发展很重要。然而，第二条基本上失活的 X 染色体如何导致女性狼疮尚不清楚。 20多年来积累的证据表明，当有遗传倾向的人暴露于导致T细胞DNA去甲基化的环境因素时，狼疮就会发生，而去甲基化的女性T细胞足以引起狼疮样自身免疫。 Richardson 实验室的工作表明，B 细胞共刺激分子 CD40L 在 X 染色体上编码，已知在狼疮 T 细胞上过度表达，但在患有狼疮的女性而非男性 T 细胞上过度表达，因为该基因位于不活跃的女性 X 染色体上。去甲基化导致双等位基因表达，而男性只能从一个基因转录。初步证据表明，女性的去甲基化 T 细胞还过度表达其他 X 染色体基因，包括趋化因子受体 CXCR3、信号调节剂 OGT 和 6 个 microRNA。基于这些观察结果，我们假设非活性 X 基因的去甲基化使女性容易患狼疮。我们将通过以下方式检验这一假设： 1. 使用高通量 DNA 测序来比较健康男性和女性 CD4+ T 细胞中 CXCR3、OGT、6 个 miRNA 基因和其他 X 染色体基因的甲基化，无论是否接受 DNA 甲基化抑制剂治疗， 2. 使用高通量 DNA 测序来比较患有狼疮的男性和女性 CD4+ T 细胞中 CXCR3、OGT、6 个 miRNA 基因和其他 X 染色体基因的甲基化情况，3.使用转染、微阵列和免疫印迹确定 OGT 过表达对 CD4+ T 细胞基因表达的影响，并使用 Argonaute Pulldown 鉴定 miRNA 靶转录本，然后进行阵列分析或测序，以及 4. 确定 CD40L、CXCR3、OGT 或 X 染色体 miRNA 是否过表达在最近开发的小鼠狼疮模型中，雄性狼疮的严重程度增加到接近女性狼疮的严重程度。我们预计这些研究将证实女性易患狼疮，部分原因是她们比男性过度表达更多的基因，识别出负责的分子，从而确定女性狼疮的新治疗靶点。 公共卫生相关性： 根据最近的估计，美国有超过 130,000 人患有 SLE。其中，近 90% 是女性，而且现在退伍军人中女性也占有相当大的比例。目前的估计显示，患有狼疮的女性每年的死亡率高达每百万人 17.6 人，这表明目前的治疗方法仍然不完善。设计更有效的治疗方法需要清楚地了解狼疮发病机制。本申请中描述的研究将描述女性易患狼疮的分子机制，从而可能有助于设计更安全、更有效的疗法来治疗这种疾病，并可能有助于治疗困扰我们女性退伍军人的其他自身免疫性疾病。